Abstract Scotopic vision is initiated upon capture of a photon of light by rhodopsin molecules present in rod photoreceptor cells. The activation of the light receptor rhodopsin sets into motion a series of biochemical reactions called phototransduction, which leads to the hyperpolarization of the cell. The long-term goal of this research program is to understand the molecular mechanisms underlying the biochemical events in phototransduction under normal and diseased states. The current focus is on rhodopsin. The importance of this molecule extends beyond its central role in phototransduction. Rhodopsin plays an important structural role and is essential for the proper formation and health of photoreceptor cells. The rhodopsin gene is a hot spot for mutations causing inherited vision disorders such as retinitis pigmentosa and congenital night blindness, which currently have no cure. Rhodopsin is a prototypical G protein-coupled receptor and therefore findings here can provide insights on other members of this superfamily of proteins that share commonalities in structure and mechanisms of action. Despite the wealth of knowledge available for rhodopsin, an accurate mechanism of its action is still unavailable and a mechanistic description on the effect of mutations in the light receptor causing vision disorders is incomplete. The current proposal is based on findings from the previous funding period that were in support of paradigms expanding on classical dogma; namely, the notion that rhodopsin forms a supramolecular structure in both healthy and diseased states and is able to achieve multiple active states, some of which may manifest only in disease. The aims of this proposal examine these paradigms in more detail and consider the implications in photoreceptor cell biology and retinal disease. In the first aim, determinants of the observed membrane organization of rhodopsin in photoreceptor cells will be characterized. In the second aim, the misfolding and aggregation of rhodopsin mutants causing retinitis pigmentosa will be characterized in cells and mouse models. In the third aim, the origin of constitutive activity in rhodopsin mutants will be characterized to better assess the mechanism of diseases such as congenital night blindness and Leber congenital amaurosis. Significant technological advances are required to overcome the intrinsic difficulties in studying membrane proteins to observe native structural and molecular details that are important to better understand the system. This proposal utilizes several innovative biophysical methods including atomic force microscopy, Frster resonance energy transfer, and Fourier transform infrared microspectroscopy. Results from our studies will lead to a more accurate mechanistic framework to understand the function of the system under normal conditions and dysfunctions in inherited retinal diseases, which will provide new avenues for scientific inquiry. The long-term impact in addressing fundamental aspects of rhodopsin structure and function will lead to the development of targeted therapeutics and discovery of novel drug targets.